CN103315985A - Application of Fingolimod in preparing medicine for inhibiting liver cancer intrahepatic metastasis - Google Patents

Abstract

The invention discloses the application of Fingolimod (code name FTY720) in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer, and belongs to the technical field of new drug applications. In the present invention, the orthotopic transplantation model of human liver cancer cells in nude mice was established, and the tumors were randomly divided into a control group and a Fingolimod treatment group. After 2 weeks, they were killed, and the tumor size and intrahepatic metastasis were recorded. It was found that Fingolimod can be transplanted in situ in liver cancer In a tumor model, it can inhibit the growth of liver cancer and reduce intrahepatic metastasis. Therefore, Fingolimod can be used to prepare drugs for inhibiting intrahepatic metastasis of liver cancer, and has good development and application prospects. The invention relates to the use of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer, which belongs to the first disclosure.

Description

Application of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer

technical field

The present invention relates to a new application of the compound Fingolimod, in particular to the application of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer. the

technical background

Fingolimod (codenamed Fingolimod), as an immunosuppressant that can inhibit lymphocyte homing, has been approved by the US FDA as an oral drug for the treatment of multiple sclerosis (MS) to reduce relapse and delay disability progression . The mechanism of action is that Fingolimod is phosphorylated in vivo and becomes an analog of S1P, which inhibits the function of S1P by competitively binding to S1PR1, and exerts its immunosuppressive and immune regulatory functions. the

During the occurrence and development of liver cancer, intrahepatic metastasis often occurs, thereby achieving evasion of immunity, radiotherapy and chemotherapy, and benefiting its own growth. Intrahepatic metastasis of liver cancer has become an important obstacle to the effective treatment and prognosis of liver cancer. Therefore, it is possible to find a cure for liver cancer The compound with inhibitory effect on internal transfer has important application value. Our study found for the first time that Fingolimod can inhibit intrahepatic metastasis of liver cancer, so Fingolimod can be used to prepare drugs for inhibiting intrahepatic metastasis of liver cancer. the

Contents of the invention

The invention provides the application of the compound Fingolimod in the preparation of a drug for inhibiting intrahepatic metastasis of liver cancer. the

The present invention adopts the following technical scheme: the application of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer. The structural formula of Fingolimod is shown in formula (I):

Formula (I)

In the present invention, the orthotopic transplantation model of human liver cancer cells in nude mice was established, and the tumors were randomly divided into a control group and a Fingolimod treatment group. After 2 weeks, they were killed, and the tumor size and intrahepatic metastasis were recorded. It was found that Fingolimod can be transplanted in situ in liver cancer In a tumor model, it can inhibit the growth of liver cancer and reduce intrahepatic metastasis. Therefore, Fingolimod can be used to prepare drugs for inhibiting intrahepatic metastasis of liver cancer, and has good development and application prospects. the

The present invention relates to the use of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer. the

The present invention will be described in further detail below through examples, but the protection scope of the present invention is not limited by any specific examples, but is defined by the claims. the

Description of drawings

Figure 1 is the establishment process of the orthotopic xenograft model of human liver cancer cells in nude mice. the

A Anesthesia and fixation; B Longitudinal laparotomy; C Exposure of liver; D Injection into tumor; E Continuous suture. the

Figure 2 is the effect of Fingolimod on liver orthotopic transplanted tumors, Figure A: control group. the

Figure 3 is the effect of Fingolimod on liver orthotopic transplanted tumors, Figure B: Fingolimod treatment group. the

Figure 4 There is a significant statistical difference between the Fingolimod treatment group and the control group. the

The tumor volume of the treatment group and the control group was statistically analyzed: there was a significant statistical difference between the two groups (P=0.006). the

Detailed ways

Compound Fingolimod involved in the present invention is purchased from R&D company. the

The present invention will be described in further detail below through examples, but the protection scope of the present invention is not limited by any specific examples, but is defined by the claims. the

Embodiment 1: the preparation of compound Fingolimod tablet involved in the present invention:

Get 20 grams of compound Fingolimod, add 180 grams of conventional excipients for tablet preparation, mix well, and make 1000 tablets with a conventional tablet press. the

Embodiment 2: the preparation of compound Fingolimod capsule involved in the present invention:

Get 20 grams of compound Fingolimod, add conventional auxiliary materials for preparing capsules such as 180 grams of starch, mix well, and pack into capsules to make 1000 tablets. the

The following pharmacodynamic experiments will further illustrate its drug activity. the

Experimental example: Establish an orthotopic transplantation model of human liver cancer cells in nude mice, and observe the inhibition of Fingolimod on liver cancer and intrahepatic metastasis in the orthotopic transplantation model of liver cancer. the

1. Materials and methods

1. Materials

1.1 Animals 12 Balb/c nude mice, male, weighing about 25g (provided by the Animal Center of Drum Tower Hospital)

1.2 Reagents

1.3 Cell lines

The human liver cancer cell line SMMC-7721 was cultured by Master Jiao Hongbo in our laboratory in Nanjing Kaiji Biotechnology Development Co., Ltd. with high-glucose DMEM medium containing 10% fetal bovine serum, and then frozen in liquid nitrogen. The human liver cancer cell line Huh7 was purchased from Xiangya School of Medicine. the

2. experimental method

2.1 Cell recovery, culture, subculture, counting

2.1.1 Cell Recovery

1. Take out the cell cryopreservation tube from the liquid nitrogen tank, and put it in the prepared 37°C water bath incubator to thaw as soon as possible. the

2. Irradiate the ultra-clean bench with ultraviolet light for 20 minutes, take out the cell suspension with a dropper and pour it into a centrifuge tube, add dropwise the high-sugar DMEM culture solution containing 10% fetal bovine serum to 10 ml, and blow gently. the

3. Centrifuge at low speed (1200 rpm) at 4°C for 5 minutes, and discard the supernatant. the

4. The centrifuged cell pellet was resuspended in high-sugar DMEM medium containing 10% fetal bovine serum by gently blowing, mixing, and cultured at 37°C in an incubator containing 95% O ₂ and 5% CO ₂ . The culture medium was discarded in the clean bench, gently washed twice with sterile PBS, and high-glucose DMEM containing 10% fetal bovine serum was added to continue the culture.

2.1.2 Cell passage

1. Discard the old culture medium when the cell density of the culture flask reaches about 90%, and gently wash twice with sterile PBS. the

2. Add an appropriate amount of 0.25% trypsin solution, discard the excess trypsin after wetting the culture bottle, place it in the incubator for 2-3 minutes, observe under a microscope, add an appropriate amount when the cells that originally adhered to the wall gradually spread out and become round The DMEM medium containing 10% fetal bovine serum and high glucose terminated the digestion, and the dropper was blown gently to make the adherent cells fall off, and then added to the centrifuge tube. the

3. Centrifuge at low speed (1200 rpm) at 4°C for 5 minutes, discard the supernatant. the

4. Add an appropriate amount of 10% fetal bovine serum high-glucose DMEM medium to resuspend gently by pipetting, then pack into culture bottles and put them in an incubator to continue culturing. the

2.1.3 Cell Count

1. After scrubbing the scoring board and cover glass with alcohol, dry them in an ultra-clean bench. the

2. Put the cover glass on the counting plate and move it slightly to one side to expose a little of the counting plate. the

3. Collect the cell pellet according to steps 1-3 of cell subculture. the

4. Add 3ml of high-sugar DMEM, and gently blow and beat the cell solution repeatedly with a dropper. the

5. Take a clean counting plate, blow the cell suspension slightly with a dropper, and immediately use a 100ml gun to absorb 20ul of the cell suspension from the edge of the cover slip to fill the gap between the counting plate and the cover slip. , no air bubbles. the

6. Observe under the microscope, the cells are required to be evenly distributed everywhere. Count the number of cells in the square grid. Those who press the center line are only counted as those on the left and above, and those on the right and below are not counted. the

7. Then calculate according to the formula: number of cells/ml = number of cells in the square grid ÷ 4×10 ⁴ ×dilution factor.

2.2 Establishment of orthotopic xenograft model of human liver cancer cells in nude mice

1. Cultivate and select SMMC-7721 cells in the logarithmic phase according to the method in the first part, trypsinize and centrifuge, and resuspend the cells in PBS so that the cell concentration is 1* ¹⁰⁸ cells/ml for later use;

2. Mix ketamine and diazepam 1:1, intramuscularly inject nude mice to anesthetize, fix on the operating table after the righting reflex disappears, connect the anesthesia maintenance pipette, prepare the skin, and routinely disinfect. the

3. Make a longitudinal incision about 1 cm below the xiphoid process in the midline of the abdomen to expose the liver, gently squeeze the bilateral ribs, squeeze out the left liver lobe, and place wet gauze under the liver lobe. the

4. Use a 1ml insulin syringe needle to stab obliquely into the liver parenchyma along the long axis of the liver lobe at an angle of 45° to the liver, and slowly inject 40 μl of SMMC-7721 cells to avoid spillage. When injecting, use a cotton swab to hold up the liver lobe, forming an angle of about 30°, which can reduce the overflow of the cell suspension. the

5. Withdraw the needle slowly and apply pressure with a sterile cotton swab for 2 to 3 minutes to prevent bleeding and reflux of the cell suspension. the

6. The liver was gently brought back into the abdominal cavity, and the abdomen was closed with layer-by-layer sutures (continuous sutures). the

7. After the operation, they were rewarmed under a warm lamp. After waking up, the mice were kept warm for 1-2 hours, and then kept in an SPF-grade environment, with free access to water and food. the

2.3 Treatment of orthotopic xenograft model of human liver cancer cells in nude mice

2.31 Experimental grouping: One month after the establishment of the orthotopic tumor model, the nude mice were randomly divided into two groups: the treatment group was injected intraperitoneally with 5 mg/(kg·day) of Fingolimod saline solution, and the control group was injected with the same amount of DMSO control intraperitoneally. , treated once a day for 14 days, and observed the activity of the nude mice. the

2.3.2 Sample collection: 14 days later, the nude mice were anesthetized and the liver was completely removed. After measuring the tumor size with a vernier caliper, each tumor was divided into two pieces, one was fixed with formaldehyde and stored at room temperature, and the other was placed in RNAlatter at 4° overnight and stored in liquid nitrogen the next day. the

2. Experimental results

1 Establishment of orthotopic xenograft model of human liver cancer cells in nude mice

In this experiment, 12 nude mice were used to establish the orthotopic tumor transplantation model, and one mouse died on the second day after operation. No tumor formation was found in anatomy, and the tumor formation rate was 92%. the

2 Treatment of orthotopic xenograft model of human liver cancer cells in nude mice

After the orthotopic transplantation model of human liver cancer in nude mice was established, they were randomly divided into two groups: Fingolimod group and control group. The results showed that all nude mice had tumors. In addition, after 14 days of intraperitoneal injection of Fingolimod at 5 mg/(kg·day), the tumor tissue was necrotic and the volume was significantly reduced (Table 1, Figure 2B). The tumor size was larger than that in the control group. There is a clear difference (Figure 3). Three cases of intrahepatic metastasis were found in the control group, and no intrahepatic metastasis was found in the Fingolimod group (Figure 2, Table 2). the

Table 1 Orthotopic tumor size in Fingolimod group and control group

Table 2 Number of intrahepatic metastases in Fingolimod group and control group

The above examples show that the present invention discloses the application of Fingolimod (code named Fingolimod) in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer, which belongs to the technical field of new applications of drugs. In the present invention, the orthotopic transplanted tumor model of human liver cancer cells in nude mice was established, and after tumor formation, they were randomly divided into a control group and a Fingolimod treatment group. After 2 weeks, they were executed, and the tumor size and intrahepatic metastasis were recorded. In the transplanted tumor model, it can inhibit the growth of liver cancer and reduce the intrahepatic metastasis (from 60% in the control group to 0%). Therefore, Fingolimod can be used to prepare drugs for inhibiting intrahepatic metastasis of liver cancer, and has good development and application prospects. The invention relates to the use of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer, which belongs to the first disclosure. the

Claims (1)

1. The application of Fingolimod in the preparation of drugs for inhibiting intrahepatic metastasis of liver cancer, the structure of the compound Fingolimod is shown in formula (I):

Formula (Ⅰ).

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